Difference between revisions of "Part:BBa K4387009"

Revision as of 16:01, 2 October 2022

Nitric Oxide Sensing Genetic Circuit Without the NorR regulator BBa_K4387001

This composite part consists of the inducible PnorV_beta, sfGFP preceded by two ribosomal binding sites and a double forward terminator. We chose a high-copy backbone from Twist for this part. Due to the competitive binding of the activated and inactivated NorR on the promoter, we decided on this construct with a positive feedback loop that adjusted the levels of NorR. The increased amount of nitric oxide in the gut activates the PnorV_beta and starts the GFP expression. When the concentration of NorR exceeds the concentration of nitric oxide, the expression of NorR is inhibited till the NO concentration is high again. In the frame of our project, we wanted to improve the sensitivity of our construct BBa_K4387005. For this purpose we removed the codon-optimized NorR, hence creating a circuit that would rely on endogenous NorR. We then tested this system in E.coli Nissle 1917. According to the data below, we could prove that the construct with two ribosomal binding sites and the presence of the codon-optimized NorR was the best and had the highest response.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal XhoI site found at 726
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

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 <partinfo>BBa_K4387009 short</partinfo>
-This composite part consists of the inducible PnorV_beta, sfGFP preceded by two ribosomal binding sites and a double forward terminator. We chose a high-copy backbone from Twist for this part. Due to the competitive binding of the activated and inactivated NorR on the promoter, we decided on this construct with a positive feedback loop that adjusted the levels of NorR. The increased amount of nitric oxide in the gut activates the PnorV_beta and starts the GFP expression. When the concentration of NorR exceeds the concentration of nitric oxide, the expression of NorR is inhibited till the NO concentration is high again. We wanted to further improve the construct BBa_K4387005 by removing the codon-optimized NorR and relying on the endogenous NorR in the E.coli Nissle 1917 genome and see if we could achieve a higher GFP response. According to the data below, we could prove that the construct with two ribosomal binding sites and the presence of the codon-optimized NorR was the best and had the highest response.
+This composite part consists of the inducible PnorV_beta, sfGFP preceded by two ribosomal binding sites and a double forward terminator. We chose a high-copy backbone from Twist for this part. Due to the competitive binding of the activated and inactivated NorR on the promoter, we decided on this construct with a positive feedback loop that adjusted the levels of NorR. The increased amount of nitric oxide in the gut activates the PnorV_beta and starts the GFP expression. When the concentration of NorR exceeds the concentration of nitric oxide, the expression of NorR is inhibited till the NO concentration is high again. In the frame of our project, we wanted to improve the sensitivity of our construct BBa_K4387005. For this purpose we removed the codon-optimized NorR, hence creating a circuit that would rely on endogenous NorR. We then tested this system in E.coli Nissle 1917. According to the data below, we could prove that the construct with two ribosomal binding sites and the presence of the codon-optimized NorR was the best and had the highest response.
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